The cellular environment is both powerful and complex, depending both on structural organization from the micron scale down to the nanometer scale, as well as on the dynamic time-dependence of a huge array of enzymes, the Nano machines of the cell, and their work on proteins and oligonucleotides. Visible fluorescence microscopy has been a useful tool capable of non-invasively exploring cellular behavior, but the limited resolution of visible light microscopy has severely restricted the information obtainable on structures on a scale below 250 nm. Because the primary bio-molecular players in cells are in the size range on the order of 10 nm, measurements are needed on this size scale in living systems. Super-resolution microscopy, either based on single-molecule fluorescence imaging and control of the emitting concentration, or on stimulated emission depletion, has solved this problem by enabling access to spatial resolutions down to the 10-40 nm regimes and below. In addition, the complementary method of single-molecule tracking provides access to the details of motions of cellular components such as the molecular motors or the motion of DNA or RNA. Combined with advanced three-dimensional (3D) imaging, single-particle tracking allows the full motion of specific cellular players to be observed in their actual context at high speed. It is a primary thrust of this work to develop and enhance both 3D super-resolution imaging and 3D single-particle tracking in cells by pushing the boundaries of both approaches and inventing new strategies to overcome critical limitations, which will lead to unprecedented spatial and temporal information in fixed and living cells. Research in the Moerner laboratory broadly addresses the limitations of super-resolution imaging and single-particle tracking in cells. A key tool involves using pupil plane modification of wide-field microscopes to provide advanced function, such as 3D imaging over unprecedented axial range or imaging of molecular orientations at the single-molecule level. The deep motivation here is to ask the fundamental question: how can the information available from each single molecule be maximized, both by measuring new variables, but also by examining every aspect of the process and inventing new methods to remove any systematic errors. The methodological developments of this research will be applied to a variety of critical problems in cell biology by continuing established collaborations and developing new collaborations with well-known biologists. The bacterium, Caulobacter crescentus, remains as a powerful model system needing elucidation of the superstructure and motions of biomolecules to understand the origins of asymmetric division. The primary cilium, a tiny but important cellular organelle, is filled with protein motions and interactions which need exploration on the nanometer scale. The organization of chromatin on all scales remains to be fully understood. These and other cell biology problems with implications for both normal and diseased function will be the focus of the application of the advanced imaging methods of this research program.